Decline in NRF2-regulated antioxidants in chronic obstructive pulmonary disease lungs due to loss of its positive regulator, DJ-1

Abstract

Rationale: Oxidative stress is a key contributor in chronic obstructive pulmonary disease (COPD) pathogenesis caused by cigarette smoking. NRF2, a redox-sensitive transcription factor, dissociates from its inhibitor, KEAP1, to induce antioxidant expression that inhibits oxidative stress.

Objectives: To determine the link between severity of COPD, oxidative stress, and NRF2-dependent antioxidant levels in the peripheral lung tissue of patients with COPD.

Methods: We assessed the expression of NRF2, NRF2-dependent antioxidants, regulators of NRF2 activity, and oxidative damage in non-COPD (smokers and former smokers) and smoker COPD lungs (mild and advanced). Cigarette smoke-exposed human lung epithelial cells (Beas2B) and mice were used to understand the mechanisms.

Measurements and main results: When compared with non-COPD lungs, the COPD patient lungs showed (1) marked decline in NRF2-dependent antioxidants and glutathione levels, (2) increased oxidative stress markers, (3) significant decrease in NRF2 protein with no change in NRF2 mRNA levels, and (4) similar KEAP1 but significantly decreased DJ-1 levels (a protein that stabilizes NRF2 protein by impairing KEAP1-dependent proteasomal degradation of NRF2). Exposure of Bea2B cells to cigarette smoke caused oxidative modification and enhanced proteasomal degradation of DJ-1 protein. Disruption of DJ-1 in mouse lungs, mouse embryonic fibroblasts, and Beas2B cells lowered NRF2 protein stability and impaired antioxidant induction in response to cigarette smoke. Interestingly, targeting KEAP1 by siRNA or the small-molecule activator sulforaphane restored induction of NRF2-dependent antioxidants in DJ-1-disrupted cells in response to cigarette smoke.

Conclusions: NRF2-dependent antioxidants and DJ-1 expression was negatively associated with severity of COPD. Therapy directed toward enhancing NRF2-regulated antioxidants may be a novel strategy for attenuating the effects of oxidative stress in the pathogenesis of COPD.

Figure 1.

Expression of NRF2 and NRF2-dependent antioxidants, and their association with lung function and protein expression in peripheral lung tissue of subjects with and without chronic obstructive pulmonary disease (COPD). (A) mRNA levels for NRF2, NQO1, GCLM, and HO1 are plotted for non-COPD and COPD lungs. The horizontal and vertical lines were plotted as median ± interquartile range. Non-COPD (n = 26) denote 8 former smokers and 11 current smokers without COPD, respectively; GOLD1–2 (n = 18) and GOLD3–4 (n = 21) represent mild and advanced COPD patient lungs, respectively. *P value < 0.001, significant with respect to non-COPD group. RFC represents the relative fold change for each mRNA expression analyzed by quantitative real-time polymerase chain reaction. GOLD = Global Initiative for Chronic Obstructive Lung Disease. (B) Spearman correlation analysis showed a significant correlation between lung function (FEV₁:FVC) and mRNA of NRF2 target antioxidants (NQO1, GCLM, and HO1). No significant correlation was observed between NRF2 mRNA and lung function. Solid triangles represent non-COPD lungs (n = 26), open squares represent GOLD1–2 lungs (n = 18), and solid dots represent GOLD3–4 lungs (n = 21). The line represents the dose–response relationship based on simple linear models on FEV₁:FVC versus log₂ mRNA levels. r = Spearman correlation coefficient. (C) Immunoblot analysis of NRF2 and its target antioxidants in lungs of subjects without COPD (n = 6) (n = 5 smokers and n = 1 ex-smoker) and subjects with advanced COPD (n = 9) (n = 6 smokers and n = 3 ex-smokers). The blots were normalized with GAPDH (glyceraldehyde phosphate dehydrogenase) as the loading control.

Figure 2.

Expression of cytosolic regulators of NRF2 in chronic obstructive pulmonary disease (COPD) and non-COPD lungs as well as association with lung function decline. (A) Quantitative real-time polymerase chain reaction (QRT-PCR) analysis of KEAP1 and DJ-1 in the lungs of patients with and without COPD. The horizontal and vertical lines were plotted as median ± interquartile range. Non-COPD (n = 26) denotes 11 former smokers and 15 current smokers without COPD, respectively; GOLD1–2 (n = 18) and GOLD3–4 (n = 21) represent mild and advanced COPD patient lungs, respectively. *P value < 0.001, significant with respect to non-COPD group. RFC represents the relative fold change for each mRNA expression analyzed by QRT-PCR. GOLD = Global Initiative for Chronic Obstructive Lung Disease. (B) Immunoblot analysis of KEAP1 and DJ-1 in the lungs of patients with and without COPD. The blots were normalized with GAPDH (glyceraldehyde phosphate dehydrogenase) as the loading control. The data are shown for six non-COPD lungs (n = 5 smokers and n = 1 ex-smoker) and nine advanced COPD lungs from GOLD3–4 patients (n = 6 smokers and n = 3 ex-smokers). (C, D) Spearman correlation analysis showed a significant correlation between lung function (FEV₁:FVC) and mRNA of NRF2 target antioxidants (NQO1, GCLM, and HO1) with DJ-1 mRNA. Solid triangles represent non-COPD control lungs (n = 26), open squares represent GOLD1–2 lungs (n = 18), and solid dots represent GOLD3–4 lungs (n = 21). The line represents the dose–response relationship based on simple linear models of FEV₁:FVC versus log₂ mRNA levels. r = Spearman correlation coefficient.

Figure 3.

Oxidative stress markers in the lungs of subjects without and with chronic obstructive pulmonary disease (COPD). (A) There was a significant decline in the levels of total glutathione (GSH) in advanced COPD lungs as compared with non-COPD lungs. (B) Levels of thiobarbituric acid–reactive substances (TBARS; marker of lipid peroxidation) were significantly elevated in advanced COPD lungs as compared with non-COPD lungs. The data were plotted as median ± interquartile range. Advanced COPD (GOLD3–4) patient lungs (n = 9) (n = 6 smokers and n = 3 ex-smokers); non-COPD control lungs (n = 6) (n = 5 smokers and n = 1 ex-smokers). *P value < 0.001, significant when compared with non-COPD lungs. GOLD = Global Initiative for Chronic Obstructive Lung Disease.

Figure 4.

Decline in NRF2-regulated antioxidant defenses in mouse lungs by DJ-1 siRNA. (A) mRNA expression of DJ-1, NRF2, and NRF2 targets NQO1 and GCLM in lungs of mice treated intratracheally with DJ-1 siRNA (20 μg/mouse in 50 μl of saline) or control siRNA (20 μg/mouse in 50 μl of saline) 5 hours after cigarette smoke exposure; n = 5. (B) Immunoblot showing the expression of DJ-1, NRF2, and NQO1 in lungs of mice treated with DJ-1 siRNA or control siRNA 5 hours after cigarette smoke exposure. GADPH (glyceraldehyde phosphate dehydrogenase) was used as the loading control. The experiments were conducted in n = 5 mice/group. The represented immunoblot shows one mouse/group. *Significant when compared with air group, P value < 0.001. CS = cigarette smoke; RFC = relative fold change.

Figure 5.

Chronic oxidative stress in Beas2B cells leads to decline in DJ-1 expression. (A) Protein expression of DJ-1 monomer and sodium dodecyl sulfate–resistant, inactive DJ-1 dimer in Beas2B cells, treated with cigarette smoke condensate (CSE; 250 μg/ml) for 6, 24, and 48 hours or dimethyl sulfoxide (DMSO). (B) Protein samples (250 μg) of Beas2B cells, treated with CSE (250 μg/ml) for 6, 24, and 48 hours or DMSO were subjected to immunoprecipitation (IP) with anti–DJ-1 antibody followed by immunoblotting (IB) with anti–2,4-dinitrophenol (DNP) antibody for detection of protein carbonyls or antioxidized DJ-1 antibody for detection of oxidized DJ-1. (D) Protein expression of DJ-1 in Beas2B cells treated with CSE (250 μg/ml) for 6, 24, and 48 hours or DMSO with or without N-acetyl cysteine (NAC; 10 mM) or MG132 (15 μM) pre- and post-treatment. The represented immunoblot shows total DJ-1 and GAPDH (glyceraldehyde phosphate dehydrogenase) as the loading control. Protein expression of NRF2 in Beas2B cells treated with CSE (250 μg/ml) for 6, 24, and 48 hours or DMSO with or without NAC (10 mM) pre- and post-treatment. The proteins were quantified using ImageJ software (National Institutes of Health, Bethesda, MD). (E) Quantization of DJ-1 relative protein expression as compared with DMSO (arbitrary units [A.U.]), for experiment shown in (D). All experiments were repeated three times.

Figure 6.

Disruption of DJ-1 in Beas2B cells and mouse embryonic fibroblasts (MEFs) impairs induction of NRF2 transcriptional activity in response to cigarette smoke condensate (CSE). (A) mRNA expression of DJ-1, NRF2, NQO1, and GCLM in Beas2B cells transfected with DJ-1 siRNA or control siRNA at 24 hours after CSE (100 μg/ml) or dimethyl sulfoxide (DMSO) treatment with or without proteasomal inhibitor MG132 (15 μM). (B) Protein expression of DJ-1, NRF2, and NQO1 in Beas2B cells transfected with DJ-1 siRNA or control siRNA at 24 hours after CSE (100 μg/ml) or DMSO treatment with or without proteasomal inhibitor MG132. The experiment was repeated three times. The represented immunoblot shows n = 2/group. (C) mRNA expression of NRF2, NQO1, and GCLM in DJ-1–deficient (DJ-1 −/−) and wild-type MEFs (DJ-1 +/+) at 24 hours after CSE (100 μg/ml) or DMSO treatment. The experiments were conducted in triplicate. (D) Antioxidant response element (ARE)–luciferase reporter activity in Beas2B cells transfected with DJ-1 siRNA or control siRNA at 24 hours after CSE or DMSO treatment with or without proteasomal inhibitor MG132. The experiments were conducted in triplicate. *Significant when compared with DJ-1–disrupted group with similar treatment, P value < 0.001; ^†significant when compared with DMSO control treatment, P value < 0.001; and ^‡significant when compared with CSE treatment, P value < 0.001. RFC = relative fold change.

Figure 7.

KEAP1 siRNA as well as sulforaphane restored the ability of DJ-1–disrupted cells to up-regulate NRF2-dependent antioxidant genes. (A) mRNA levels of NQO1 and GCLM in DJ-1–deficient and wild-type mouse embryonic fibroblasts (MEFs) after 48 hours of KEAP1 siRNA or control siRNA transfection with or without cigarette smoke condensate (CSE) stimulus. After 36 hours of siRNA transfection, cells were challenged with CSE for 12 hours. (B) Sulforaphane (5 μM) induced mRNA expression of NQO1 and GCLM in Beas2B cells transfected with DJ-1 siRNA or control siRNA with or without CSE treatment. After 36 hours of transfection, cells were treated with CSE for 12 hours followed by sulforaphane for an additional 12 hours. The experiments were conducted in triplicate. mRNA was measured 12 hours after sulforaphane treatment. (C) Sulforaphane induced mRNA expression of NQO1 and GCLM in DJ-1–deficient and wild-type MEFs with or without CSE treatment. At 12 hours after CSE or vehicle treatment, cells were challenged with or without sulforaphane (5 μM) and incubated for an additional 12 hours. *Significant when compared with the DJ-1–disrupted group with similar treatment, P value < 0.001; ^†significant when compared with dimethyl sulfoxide (DMSO) treatment, P value < 0.001; and ^‡significant when compared with CSE treatment, P value < 0.001. RFC = relative fold change.

Figure 8.

Schematic showing working model for DJ-1–mediated regulation of the NRF2 pathway. ARE = antioxidant response element; ROS = reactive oxygen species.